Fusion protein and medical application thereof

Abstract

The present disclosure provides fusion proteins comprising a modified spacer domain that increases antigen binding affinity, antibodies against CD19 and their use in the treatment of disease.

Description

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to international patent applications with application numbers PCT / CN2023 / 099418 and PCT / CN2024 / 091948, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure generally relates to fusion proteins containing modified hinge domains and methods of using them. Background Technology

[0004] Chimeric antigen receptor (CAR) T cells have demonstrated significant response rates in patients with certain subtypes of B-cell leukemia or lymphoma, and encouraging results have also been observed in patients with multiple myeloma. CAR constructs typically comprise an extracellular antigen-binding domain, a spacer region, a transmembrane domain, and an intracellular signaling domain. The spacer region, located between the extracellular antigen-binding domain and the transmembrane domain, plays a crucial role in mediating antitumor activity. CARs with improved spacer region domains are needed. Summary of the Invention

[0005] This disclosure provides novel fusion proteins comprising modified spacer domains, which possess properties advantageous for cell therapy. Specifically, this disclosure relates to CARs having modified spacer domains that allow the CAR to retain stabilization capabilities, maintain the conformation of the antigen-binding domain within the CAR, and / or position the antigen-binding domain at a distance from the target epitope, said distance enabling the CAR to effectively interact with target cells.

[0006] In a first aspect, this disclosure provides a fusion protein comprising a) an extracellular antigen-binding domain; b) a spacer region consisting of the sequence of SEQ ID NO: 28 or a functional equivalent thereof; c) a transmembrane domain; and d) an intracellular signal transduction domain.

[0007] This article also provides a fusion protein wherein the spacer region consists of the amino acid sequence of SEQ ID NO: 31 (EX1KSCDTPPPX2PX3CP); wherein X1 is S or P; X2 is C or S; and X3 is R or P. In one aspect, the spacer region consists of the amino acid sequence of SEQ ID NO: 31 (EX1KSCDTPPPX2PX3CP); wherein X1 is S; X2 is C or S; and X3 is R or P. In one aspect, the spacer region consists of the amino acid sequence of SEQ ID NO: 28.

[0008] This article also provides a fusion protein wherein the extracellular antigen-binding domain comprises an antibody containing a heavy chain variable region (VH) and a light chain variable region (VL). In one aspect, the antibody is capable of binding to the same epitope on CD19 with a reference antibody containing the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 25. In one aspect, VH comprises HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; and VL comprises LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively. In one aspect, VH comprises HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; VL comprises LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

[0009] This document also provides a fusion protein, wherein VH comprises the amino acid sequence of SEQ ID NO: 1; VL comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2, wherein the amino acids at positions 39 and 80 of VL are R and P, respectively. In one aspect, VL comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 2, wherein the amino acids at positions 39 and 80 of VL are R and P, respectively. In one aspect, the amino acids at positions 100 and 103 of VL are S and R, respectively. In one aspect, the amino acids at positions 39, 80, 100, and 103 of VL are R, P, S, and R, respectively. In one aspect, VH comprises the amino acid sequence of SEQ ID NO: 1; VL comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

[0010] This article also provides a fusion protein wherein the antibody is scFv. In one aspect, VH is fused to the C-terminus of VL via a flexible peptide linker at its N-terminus. In one aspect, the flexible peptide linker comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20. In one aspect, scFv comprises the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In one aspect, scFv comprises the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In one aspect, scFv comprises the amino acid sequence of SEQ ID NO: 22.

[0011] This article also provides a fusion protein wherein the transmembrane domain comprises a transmembrane domain of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, or PD1. In one aspect, the transmembrane domain comprises a transmembrane domain of CD8α, CD4, or CD28. In another aspect, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 32.

[0012] This article also provides a fusion protein wherein an intracellular signal transduction domain comprises a co-stimulatory signal transduction domain and / or a primary intracellular signal transduction domain. In one aspect, the co-stimulatory signal transduction domain is fused to the N-terminus of the primary intracellular signal transduction domain via its C-terminus. In one aspect, the co-stimulatory signal transduction domain is a signal transduction domain selected from ligands of CD27, CD28, CD137, OX40, CD30, CD40, CD3, HVEM, ICOS, Myd88, LFA-1, ICOS, CD2, CD7, NKG2C, B7-H3, and CD83, and combinations thereof. In one aspect, the co-stimulatory signal transduction domain is a CD28 or CD137 signal transduction domain. In one aspect, the co-stimulatory signal transduction domain is a CD137 signal transduction domain. In one aspect, the primary intracellular signal transduction domain comprises a cytoplasmic signal transduction domain of CD3ζ. In one aspect, the co-stimulatory signal transduction domain is a CD28 or CD137 signal transduction domain; the primary intracellular signal transduction domain includes the cytoplasmic signal transduction domain of CD3ζ.

[0013] This article also provides a fusion protein, wherein the fusion protein is a CAR, and the CAR comprises, from its N-terminus to its C-terminus, an extracellular antigen-binding domain, a spacer region, a transmembrane domain, and an intracellular signal transduction domain. In one aspect, the extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23, and the spacer region comprises the amino acid sequence of SEQ ID NO: 28. In one aspect, the CAR comprises the amino acid sequence of SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37. In another aspect, the CAR comprises the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

[0014] A second aspect of this disclosure provides an antibody binding to CD19, comprising VH and VL, wherein VH comprises the sequence of SEQ ID NO: 1; and VL comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 2, wherein amino acids at positions 39 and 80 of VL are R and P, respectively. In one aspect, VL comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 2, wherein amino acids at positions 39 and 80 of VL are R and P, respectively. In one aspect, amino acids at positions 100 and 103 of VL are S and R, respectively. In one aspect, amino acids at positions 39, 80, 100, and 103 of VL are R, P, S, and R, respectively. In one aspect, VH comprises the amino acid sequence of SEQ ID NO: 1; and VL comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

[0015] This article also provides an antibody, wherein the antibody is scFv. In one aspect, VH is fused to the C-terminus of VL via a flexible peptide linker at its N-terminus. In one aspect, the flexible peptide linker comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20. In one aspect, scFv comprises the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In one aspect, scFv comprises the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In one aspect, scFv comprises the amino acid sequence of SEQ ID NO: 22.

[0016] This disclosure also provides a complex comprising an antibody and a heterologous molecule or portion thereof as described herein according to the disclosure. In one aspect, the heterologous molecule or portion is covalently bound to the antibody. In another aspect, the complex is a fusion protein comprising, from its N-terminus to its C-terminus, an extracellular antigen-binding domain comprising the antibody, a transmembrane domain, and an intracellular signal transduction domain.

[0017] This disclosure also provides a nucleic acid encoding an isolated fusion protein or antibody as described herein according to the disclosure.

[0018] This disclosure also provides a vector comprising nucleic acids as described herein according to this disclosure. In one aspect, the vector is a viral vector. In another aspect, the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus vector.

[0019] This disclosure also provides a cell comprising a fusion protein, antibody, complex, isolated nucleic acid, or vector as described herein according to the disclosure. In one aspect, the cell is a lymphocyte. In another aspect, the cell is an NK cell or a T cell.

[0020] This disclosure also provides a pharmaceutical formulation comprising a fusion protein, antibody, complex, or cell as described herein, and a pharmaceutically acceptable carrier.

[0021] This disclosure also provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 31 (EX1KSCDTPPPX2PX3CP), wherein X1 is S or P; X2 is C or S; and X3 is R or P. In one aspect, the polypeptide is contained in a fusion protein. In one aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 31 (EX1KSCDTPPPX2PX3CP); wherein X1 is S; X2 is C or S; and X3 is R or P. In one aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 28.

[0022] This disclosure also provides a method of treating cancer or an autoimmune disease in an individual, comprising administering to the individual a fusion protein, antibody, complex, cell, or pharmaceutical preparation as described herein according to this disclosure. Use of the fusion protein, antibody, complex, cell, or pharmaceutical preparation as described herein according to this disclosure in the preparation of a medicament for treating cancer or an autoimmune disease is also provided. This disclosure further provides a fusion protein, antibody, complex, cell, or pharmaceutical preparation as described herein according to this disclosure, which is used as a medicament. In one aspect, the cancer is selected from leukemia, lymphoma, lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, and rhabdomyosarcoma. In one aspect, the cancer is leukemia or lymphoma. In one aspect, the autoimmune disease is selected from systemic lupus erythematosus, lupus nephritis, multiple sclerosis, rheumatoid arthritis, Sjögren's syndrome, idiopathic thrombocytopenic purpura, type 1 diabetes mellitus, pemphigus vulgaris, neuromyelitis optica, ANCA vasculitis, and myasthenia gravis. One aspect is that autoimmune diseases include systemic lupus erythematosus or lupus nephritis. Attached Figure Description

[0023] Figures 1A to 1C Display the binding constants (K) of FMC63-scFv, scFv-1, scFv-2, and scFv-3 respectively. a ), binding rate (K) on ) and dissociation rate (K off ).

[0024] Figure 2 The binding affinity of CAR-W and CAR-1 to CAR-12 on T cells for CD19 was demonstrated.

[0025] Figure 3 This involves tonic signaling in CAR-W and CAR-1 through CAR-12.

[0026] Figures 4A to 4C The activity of the CAR containing the spacer region S-3 was shown. Figure 4A The expression levels of CD19 in different target cells were shown. Figure 4B This demonstrates the lethality of CAR. Figure 4C The study showed CD69 expression on CAR-induced T cells.

[0027] Figures 5A to 5D It involves the activation and expansion of T cells after target cell stimulation. Figure 5A , 5B 5C showed IFN-γ, TNF-α, and IL-2 secreted by CAR-transduced T cells. Figure 5D The study showed T cell proliferation 7 days after initial stimulation.

[0028] Figure 6 The study showed CAR-T cell killing of Raji-CD19Lo target cells on day 3 of co-culture assays (E:T=1:1, 1:2, 1:8). Detailed Implementation

[0029] I. Definition

[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0031] The singular forms of the terms ("a" and "an") refer to one or more (i.e., at least one) grammatical objects of the article. For example, "component" means one or more components.

[0032] As used herein, the terms “comprising, comprises” are synonymous with “including, includes” or “containing”, and are inclusive or open-ended, and do not exclude additional, unlisted members, elements, or methodological steps. The terms “comprising, comprises” and the like also include the term “consisting of”.

[0033] The term “antibody” is used in the broadest sense herein and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), intact antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0034] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably in this document and refer to antibodies with a structure substantially similar to that of natural antibodies. For example, an intact IgG antibody comprises two light chains and two heavy chains linked by disulfide bonds. Each heavy chain has a variable domain (VH) from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3), also known as the heavy chain constant region. Similarly, each light chain has a variable domain (VL) from the N-terminus to the C-terminus, followed by a constant light chain (CL) domain, also known as the light chain constant region. Antibody heavy chains can be classified into one of five types, referred to as IgA, IgD, IgE, IgG, or IgM, some of which can be further subdivided into subtypes such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Based on the amino acid sequence of the constant domains, antibody light chains can be classified into one of two types, referred to as kappa (κ) and lambda (λ).

[0035] "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of a complete antibody that binds to an antigen. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, linear antibodies, single-chain antibody molecules (such as scFv and scFab), single-domain antibodies, and multispecific antibodies formed from antibody fragments.

[0036] "Single-chain Fv" or "scFv" is a fusion protein of the variable regions of the light chain (VL) and heavy chain (VH), linked by a short linker peptide of approximately 10-25 amino acids. The linker is typically flexible and can connect the N-terminus of the VH to the C-terminus of the VL, and vice versa. Despite the removal of the constant region and the introduction of the linker, this protein retains the specificity of the original antibody.

[0037] The term "antigen-binding domain" refers to an antibody portion containing a region that binds to and is complementary to some or all of an antigen. The antigen-binding domain can be provided, for example, by one or more antibody variable domains (also called antibody variable regions). In a preferred aspect, the antigen-binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0038] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable regions (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and complementarity-determining regions (CDRs). A single VH or VL domain is sufficient to confer antigen-binding specificity. Furthermore, VH or VL domains can be used to isolate antibodies binding to specific antigens from antibodies binding to antigens, allowing for screening of libraries containing complementary VL or VH domains, respectively. As used herein, the "Kabat number" associated with the variable region sequence refers to the numbering system proposed by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0039] The terms “complementarity-determining region” and “CDR” are known to refer to the discontinuous amino acid sequence within an antibody variable region that confers antigen specificity and / or binding affinity. Typically, three CDRs (HCDR1, HCDR2, HCDR3) are present in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) are present in each light chain variable region. “Frame region” and “FR” refer to the non-CDR portions of the heavy and light chain variable regions. Table 1 below lists exemplary positional boundaries of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as determined by the Kabat, Chothia, AbM, IMGT, and Contact protocols, respectively. Unless otherwise stated, the amino acid sequences of the CDRs shown in this disclosure are determined according to Kabat.

[0040] Table 1. CDR areas according to various numbering systems

[0041] The term "humanized" antibody refers to a chimeric antibody comprising amino acid residues from a non-human CDR and amino acid residues from a human framework. In some embodiments, the humanized antibody will comprise at least one (typically two) variable domains, wherein all or substantially all of the CDRs correspond to the CDRs of the non-human antibody, and all or substantially all of the FRs correspond to the FRs of the human antibody.

[0042] The term "human" antibody refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source utilizing a human antibody library or other human antibody-coding sequences. The definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0043] As used herein, the interchangeable terms “specific binding” or “binding” mean the ability of a protein to bind to a target protein under specific binding conditions such that its affinity or affinity is at least 5 times, but optionally at least 10, 20, 30, 40, 50, 100, 250, or 500 times, or even at least 1000 times, the average affinity or affinity of the same protein for a statistically significant set of random peptides or polypeptides. Specific binding proteins do not need to bind only a single target molecule, but can specifically bind non-target molecules because of the structural conformational similarity between the target and non-target molecules (e.g., paralogs or orthologs). Those skilled in the art will recognize that specific binding with molecules that have the same function in different animal species or with non-target molecules having epitopes substantially similar to those of the target molecule is possible and does not diminish the specificity of the binding, which is determined relative to a statistically valid set of unique non-target molecules. Therefore, due to cross-reactivity, polypeptides can specifically bind to more than one different kind of target molecule.

[0044] The term "epitope" refers to an antigenic moiety that specifically interacts with an antibody. Such a moiety (referred to herein as an epitope determinant) typically contains or is part of an element such as an amino acid side chain or a sugar side chain. Epitope determinants can be defined, for example, by methods known in the art, such as crystallography or by hydrogen-deuterium exchange. At least one or more of the moiety on the antibody molecule that specifically interacts with the epitope determinant is typically located in the CDR. Generally, epitopes have specific three-dimensional structural features and / or specific charge features. Some epitopes are linear epitopes, while others are conformational epitopes.

[0045] The term "hinge" refers to the portion of an antibody heavy chain polypeptide that connects the CH1 and CH2 domains in a wild-type antibody heavy chain. In this disclosure, the hinge contained in the fusion protein comprises a naturally occurring or modified sequence.

[0046] The "percentage of amino acid sequence identity (%)" and "homology" relative to peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, after sequence alignment and the introduction of vacancies (if necessary) to obtain the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. To determine the percentage of amino acid sequence identity, alignment can be performed using various methods known to those skilled in the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0047] The term "fragment" (e.g., hinge) of a polypeptide refers to an amino acid sequence that is shorter than the naturally occurring sequence, and that lacks the N-terminus and / or C-terminus, or any part of the polypeptide, compared to the naturally occurring polypeptide. Therefore, a fragment does not necessarily need to be missing only the N-terminal and / or C-terminal amino acids. Polypeptides in which internal amino acids are missing relative to the naturally occurring sequence are also considered fragments. The term "C-terminal fragment" refers to a fragment that has the N-terminal amino acid missing only from the naturally occurring polypeptide.

[0048] The term "functional equivalent" refers to a polypeptide that retains the function of the polypeptide. Thus, in some respects, hinged functional equivalents retain the ability to stabilize CAR molecules, maintain the conformation and valence state of the antigen-binding domain (e.g., scFv) in the CAR, and / or position the antigen-binding domain at a distance from the target epitope that allows the CAR to interact effectively with the target cell.

[0049] The term "fusion protein" refers to a hybrid (e.g., chimeric, recombinant) polypeptide that contains protein domains from at least two different proteins.

[0050] The term "chimeric antigen receptor" or "CAR" refers to a fusion protein engineered to comprise two or more naturally occurring or engineered amino acid sequences linked together in a manner not naturally present in host cells, which, when present on the cell surface, can act as a receptor. The CAR of this disclosure includes an extracellular portion comprising an antigen-binding domain (e.g., scFv) linked to a spacer region sequence, a transmembrane domain, and one or more intracellular signaling domains (optionally containing a co-stimulatory domain).

[0051] As used herein, the interchangeable terms "extracellular domain" or "ectodomain" refer to a region of a membrane protein (e.g., a transmembrane protein) located outside the vesicle membrane. An extracellular domain typically contains binding domains that specifically bind ligands or cell surface receptors, for example, binding domains that specifically bind ligands or cell surface receptors. The term "extracellular antigen-binding domain" refers to an extracellular domain or a portion of an extracellular domain capable of specifically binding antigens.

[0052] As used herein, the interchangeable terms “endodomain” or “intramidal domain” or “cytoplasmic domain” refer to regions found in some membrane proteins (e.g., transmembrane proteins) that extend into the internal space defined by the cell surface membrane. In some cells, endodomains interact with intracellular components and may play a role in signal transduction, and therefore may be intracellular signal transduction domains in some cases.

[0053] As used in this article, the term "transmembrane domain" refers to a domain found in membrane proteins that essentially or completely spans the lipid bilayer, such as those found in biological membranes (e.g., mammalian cells) or in artificial constructs (e.g., liposomes). Transmembrane proteins can cross both layers of the lipid bilayer once or multiple times.

[0054] As used herein, the term "spacer region" refers to a polypeptide sequence that can covalently link two spacer portions together: the antigen-binding domain and the transmembrane domain of a fusion protein (e.g., CAR).

[0055] As used in this article, the term "flexible peptide linker" refers to a peptide that connects other peptides to form a functional protein. In the context of scFv, a flexible peptide linker refers to a peptide linker that connects VH and VL to form an scFv that specifically binds to the antigen.

[0056] The term "self" refers to any material that comes from the same individual and is subsequently introduced back into that individual.

[0057] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material was introduced. Two or more individuals are called allologous to each other when the genes at one or more loci are not identical. In some respects, allologous material from individuals of the same species may be genetically sufficiently different to interact antigenically.

[0058] The term "expression" refers to the process by which a polypeptide is produced based on the coding sequence of a nucleic acid molecule, such as a gene. This process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

[0059] The term "encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes, which have defined nucleotide sequences (e.g., rRNA, tRNA, and mRNA) or defined amino acid sequences, and the resulting biological properties. Therefore, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, then that gene, cDNA, or RNA encodes that protein.

[0060] The terms "polypeptide," "peptide," and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise a peptide sequence. The term "polypeptide" also means a product of post-expression modification of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. The polypeptides of this disclosure can be of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such a structure.

[0061] "Isolated" nucleic acid molecules refer to nucleic acid molecules that have been isolated from components of their natural environment. Isolated nucleic acid molecules include those normally found in cells that contain them, but which are located extrachromosomally or at chromosomal locations different from their natural chromosomal locations. "Isolated polynucleotides (or nucleic acids) encoding fusion proteins" refers to one or more polynucleotide molecules encoding fusion proteins, including such polynucleotide molecules in a single vector or different vectors, and such polynucleotide molecules present at one or more locations within host cells.

[0062] As used herein, the term "vector" refers to a nucleic acid molecule capable of proliferating another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which they have been introduced. Some vectors can direct the expression of nucleic acids to which they are operatively linked.

[0063] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of these cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of passage number. The nucleic acid content of progeny cells may not be exactly the same as that of parent cells, but may contain mutations. This document includes mutant progeny that have the same function or biological activity as those screened or selected in the original transformed cells. Host cells are any type of cell system that can be used to produce the antibodies of this invention. Host cells include cultured cells, such as mammalian cultured cells like HEK cells, CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, to name just a few, but also include cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues. In one aspect, the host cells of this invention are eukaryotic cells, particularly mammalian cells. In another aspect, the host cells are not cells within the human body.

[0064] The term "subject" is intended to include living organisms (e.g., mammals, humans) in which an immune response can be elicited.

[0065] As used herein, “treatment” (and its grammatical variations, such as “treat” or “treating”) refers to a clinical intervention that attempts to alter the natural course of disease in the individual being treated, and may be used for prevention or during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, relieving symptoms, mitigating any direct or indirect pathological consequences of disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.

[0066] The term "pharmaceutical composition" refers to an article in a form in which the biological activity of the active ingredient contained therein is effective, and which does not contain any other components that would have unacceptable toxicity to the individual to whom the article will be administered. Pharmaceutical compositions typically contain one or more pharmaceutically acceptable carriers. "Pharmaceutically acceptable carrier" refers to a component of the pharmaceutical composition that is not toxic to the individual, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0067] The “effective amount” of an active agent (such as a pharmaceutical composition) refers to the amount that effectively achieves the desired therapeutic or preventative outcome within the necessary dosage and time period.

[0068] II. Fusion Proteins

[0069] This disclosure provides a novel fusion protein with advantageous properties such as manufacturability, stability, binding affinity, bioactivity, targeting efficiency, reduced toxicity, an extended dose range for patient administration (e.g., contained in cells), and potentially enhanced efficacy. The novel fusion protein includes a spacer region that improves antigen binding, CAR stability, and the ability to kill target cells expressing low-density ligands.

[0070] This document provides a fusion protein comprising a) an extracellular antigen-binding domain; b) a spacer region consisting of the amino acid sequence of SEQ ID NO: 28 or its functional equivalent; c) a transmembrane domain; and d) an intracellular signal transduction domain. In some embodiments, the fusion protein is a CAR.

[0071] 2.1 Interval Zone

[0072] In one aspect, fusion proteins as defined herein are provided, wherein a fusion protein comprising a spacer region consisting of a functional equivalent of SEQ ID NO: 28 exhibits a difference in CD19 binding value of no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% compared to a fusion protein comprising a spacer region consisting of SEQ ID NO: 28.

[0073] In one aspect, the spacer region comprises at least a portion of an immunoglobulin constant region or a variant or modified form thereof, such as a hinge region. In one aspect, the spacer region comprises at least a portion of an IgG hinge. In one aspect, the spacer region may be a chimeric polypeptide containing one or more hinge fragments derived from IgG1, IgG2, IgG3, and / or IgG4. In one aspect, the spacer region may be a chimeric polypeptide containing one or more hinge fragments derived from IgG3. In one aspect, the spacer region consists of an amino acid sequence of 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues in length. In some embodiments, the spacer region consists of an amino acid sequence of 13-17 amino acid residues in length. In a preferred embodiment, the spacer region consists of an amino acid sequence of 15 amino acid residues in length. In one aspect, the spacer region consists of an amino acid sequence that is at least about 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 28.

[0074] In one aspect, the spacer region consists of the amino acid sequence of SEQ ID NO: 31 (EX1KSCDTPPPX2PX3CP); wherein X1 is S or P; X2 is C or S; and X3 is R or P. In one aspect, X1 is S; X2 is C or S; and X3 is R or P. In one aspect, X1 is P; X2 is C or S; and X3 is R or P. In one aspect, X1 is S or P; X2 is C; and X3 is R or P. In one aspect, X1 is S or P; X2 is C or S; and X3 is R. In one aspect, X1 is S or P; X2 is C or S; and X3 is P. In one aspect, X1 is S; X2 is C; and X3 is R or P. In one aspect, X1 is S; X2 is S; and X3 is R or P. In one aspect, X1 is S; X2 is C; and X3 is R. In one aspect, X1 is S; X2 is C; and X3 is P. In one aspect, X1 is S; X2 is S; and X3 is R. In one aspect, X1 is S; X2 is S; and X3 is P. In one specific embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO: 28.

[0075] 2.2 Extracellular antigen-binding domain

[0076] In one aspect, a fusion protein as defined earlier herein is provided, wherein the extracellular antigen-binding domain comprises one or more antibodies. In another aspect, the antibody is Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, linear antibody, single-chain antibody molecule (e.g., scFv and scFab), single-domain antibody, or multispecific antibody formed from antibody fragments. In a specific embodiment, the extracellular antigen-binding domain comprises scFv.

[0077] In one aspect, the extracellular antigen-binding domain is capable of binding tumor antigens or pathogen antigens. In another aspect, the tumor antigen is a tumor-specific antigen or tumor-associated antigen (TAA). Various TAAs are known, some of which are shown in Table 2 below. The antigen-binding domain used in this disclosure is a domain capable of binding TAAs as shown therein.

[0078] Table 2. TAA Types

[0079] In one aspect, the tumor antigen is CD19. In one aspect, the fusion protein is capable of binding to the same epitope on CD19 with any anti-CD19 antibody in the art (e.g., FMC63, SC25C1). In one aspect, the extracellular antigen-binding domain of the fusion protein comprises scFv, which is capable of binding to the same epitope on CD19 with a reference antibody comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 25. In one aspect, scFv comprises VH and VL, VH comprising HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; and VL comprising LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively. In one aspect, scFv comprises VH and VL, VH comprising HCDR1, HCDR2 and HCDR3 containing the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 respectively; VL comprising LCDR1, LCDR2 and LCDR3 containing the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 respectively.

[0080] In one aspect, scFv comprises VH and VL, wherein VH comprises the amino acid sequence of SEQ ID NO: 1; and VL comprises the amino acid sequence of SEQ ID NO: 2. In one aspect, VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO: 1. In one aspect, VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO: 2. In one aspect, VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO: 2, wherein the amino acids at positions 39 and 80 of VL are R and P, respectively. In one aspect, VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, or 96% sequence identity with SEQ ID NO: 2, wherein the amino acids at positions 39, 80, 100, and 103 of VL are R, P, S, and R, respectively. In a specific embodiment, VH comprises or is composed of the amino acid sequence of SEQ ID NO: 1; VL comprises or is composed of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

[0081] In one aspect, the scFv comprises a humanized antibody or a human antibody VH and VL. Exemplary scFvs are described in patent numbers WO2010095031, WO2014153270, WO2017015783, CN107383196A, CN111848801A, WO2018200496, or WO2022105811.

[0082] In one aspect, VH and VL are linked via a flexible peptide linker. In another aspect, VH is fused to the C-terminus of VL via a flexible peptide linker through its N-terminus (LH). In another aspect, VL is fused to the C-terminus of VH via a flexible peptide linker through its N-terminus (HL). In one aspect, the flexible peptide linker is (G4S). n (SEQ ID NO:42), (SG4) n (SEQ ID NO:43) or G4 (SG4) n(SEQ ID NO:44), where “n” is 1, 2, 3, 4, 5, 6, 7, or 8, particularly 3. In one aspect, the flexible polypeptide linker comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20. In one aspect, the ScFv comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25. In one aspect, the ScFv comprises the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25. Table 3 provides SEQ ID NOs of exemplary scFvs.

[0083] Table 3. SEQ ID NO of exemplary scFv

[0084] 2.3 Transmembrane domain

[0085] In one aspect, fusion proteins as defined herein are provided, wherein the transmembrane domain can be obtained from naturally occurring proteins or synthetic, non-naturally occurring protein segments, such as thermodynamically stable hydrophobic protein segments in the cell membrane. In another aspect, the protein segment is at least about 20 amino acids, for example, at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids.

[0086] In one aspect, the transmembrane domains provided herein are derived from type I single-pass membrane proteins. In another aspect, transmembrane domains derived from multi-pass membrane proteins are also compatible with the fusion proteins described herein. Multi-pass membrane proteins may comprise complex (at least 2, 3, 4, 5, 6, 7 or more) α-helical or β-sheet structures. In one aspect, the N-terminus and C-terminus of a multi-pass membrane protein are located on opposite sides of a lipid bilayer; for example, the N-terminus of the protein is located on the cytoplasmic side of the lipid bilayer, and the C-terminus of the protein is located on the extracellular side.

[0087] In one aspect, the transmembrane domain provided herein may comprise a transmembrane region and a cytoplasmic region located on the C-terminal side of the transmembrane domain. The cytoplasmic region of the transmembrane domain may contain three or more amino acids, and in some embodiments, facilitates the orientation of the transmembrane domain within a lipid bilayer. In one aspect, one or more cysteine ​​residues are present in the transmembrane region of the transmembrane domain. In one aspect, one or more cysteine ​​residues are present in the cytoplasmic region of the transmembrane domain. In one aspect, the cytoplasmic region of the transmembrane domain contains positively charged amino acids. In one aspect, the cytoplasmic region of the transmembrane domain contains the amino acids arginine, serine, and lysine.

[0088] In one aspect, the transmembrane region of the transmembrane domain contains hydrophobic amino acid residues. In another aspect, the transmembrane domain of the CAR provided herein contains an artificial hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan, and valine may be present at the C-terminus of the transmembrane domain. In one aspect, the transmembrane region primarily contains hydrophobic amino acid residues such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In one aspect, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region contains a polyleucine-alanine sequence.

[0089] In one aspect, the transmembrane domain comprises transmembrane domains selected from: the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, GPC3, IL-2Rβ, IL-2R γ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, I TGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In one aspect, the transmembrane domain is derived from or comprises transmembrane domains of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some specific embodiments, the transmembrane domain comprises an amino acid sequence having at least 88%, 92%, and 96% sequence identity with SEQ ID NO: 32. In specific embodiments, the transmembrane domain comprises or is composed of the amino acid sequence of SEQ ID NO: 32.

[0090] 2.4 Signal Transduction Structural Domain

[0091] In one aspect, fusion proteins as defined herein are provided, wherein an intracellular signaling domain is typically responsible for activating at least one normal effector function. In another aspect, the intracellular signaling domain comprises one or two different types of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via a TCR (primary intracellular signaling domains) and / or those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic domains, such as co-stimulatory domains).

[0092] In one aspect, the primary signaling domain regulates primary activation of the TCR complex in a stimulatory or inhibitory manner. The primary intracellular signaling domain acting in a stimulatory manner may contain a signaling motif, referred to as an immune receptor tyrosine-based activation motif (ITAM). Examples of ITAM-containing primary intracellular signaling domains particularly useful in this invention include those of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FceRI, DAP10, DAP12, and CD66d. In one aspect, the CAR comprises an intracellular signaling domain, such as the primary signaling domain of CD3-ζ. In one aspect, the primary signaling domain comprises a modified ITAM domain, for example, a mutated ITAM domain whose activity has been altered (e.g., increased or decreased) compared to the native ITAM domain. In one aspect, the primary signal transduction domain comprises a modified ITAM-containing primary intracellular signal transduction domain, such as an optimized and / or truncated ITAM-containing primary intracellular signal transduction domain. In one aspect, the CD3-ζ signal transduction domain is a mutant CD3ζ or wild-type human CD3ζ. In one aspect, the primary signal transduction domain comprises one, two, three, four, or more ITAM motifs. In one aspect, the primary intracellular signal transduction domain is a functional mutant of the cytoplasmic signal transduction domain of CD3ζ containing one or more mutations (e.g., Q65K).

[0093] In one aspect, intracellular signaling sequences within the cytoplasm can be linked together in a random or designated order. In another aspect, co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for an effective lymphocyte response to antigens. Examples of such molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, signaling lymphocyte activating molecules (SEAM proteins), activating NK cell receptors, BTFA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, FFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, FIGHT, HVEM, KIRDS2, SFAMF7, and NKp80. (KFRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IF2Rβ, IF2Rγ, IF7Rα, ITGA4, VFA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VFA-6, CD49f, ITGAD, CD 11d, ITGAE, CD103, ITGAF, FFA-l, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, FFA-l, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKF, DNAM1 (CD226), SFAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Fy9 (CD229), CD160 (BY55), PSGF1, CD100 (SEMA4D), CD69, SFAMF6 (NTB-A, Fyl08), SEAM (SFAMF1, CD150, IPO-3), BFAME (SFAMF8), SEFPFG (CD162), FTBR, FAT, GADS, SFP-76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83, etc.

[0094] In one respect, the costimulatory signaling domain contains up to 10 amino acid residue variations (e.g., 1, 2, 3, 4, 5, or 8) compared to its wild-type counterpart. Such a costimulatory signaling domain containing one or more amino acid variations may be referred to as a variant. Mutations in amino acid residues of the costimulatory signaling domain, relative to the non-mutated domain, can lead to increased signal transduction and enhanced stimulation of the immune response. Conversely, mutations in amino acid residues of the costimulatory signaling domain, relative to the non-mutated domain, can lead to decreased signal transduction and reduced stimulation of the immune response.

[0095] In one aspect, one or more costimulatory signaling domains are selected from costimulatory signaling domains of CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, NKG2C, B7-H3, and ligands that specifically bind to CD83. In one aspect, the intracellular signaling domain in the CAR of this disclosure comprises a costimulatory signaling domain derived from CD137 (4-1BB). In one aspect, the intracellular signaling domain comprises a cytoplasmic signaling domain of CD3ζ and a costimulatory signaling domain of CD28 or CD137. In a specific embodiment, the intracellular signaling domain comprises a cytoplasmic signaling domain of CD3ζ and a costimulatory signaling domain of CD137.

[0096] 2.5 CAR

[0097] In one aspect, a fusion protein as defined herein is provided, wherein the fusion protein is a CAR comprising, from its N-terminus to its C-terminus, an extracellular antigen-binding domain, a spacer region, a transmembrane domain, and an intracellular signal transduction domain. In one aspect, the extracellular antigen-binding domain is a scFv comprising, from its N-terminus to its C-terminus, a VL, a linker, and a VH. In one aspect, the extracellular antigen-binding domain is a scFv comprising or consisting of the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In one aspect, the spacer region comprises the amino acid sequence of SEQ ID NO: 28. In one aspect, the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 32. In one aspect, the intracellular signal transduction domain comprises, from its N-terminus to its C-terminus, a co-stimulatory signal transduction domain and a primary intracellular signal transduction domain. In one aspect, the intracellular signal transduction domain comprises, from its N-terminus to its C-terminus, a signal transduction domain of CD28 or CD137 and a cytoplasmic signal transduction domain of CD3ζ. In one specific embodiment, the CAR comprises or consists of the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

[0098] III. Antibodies

[0099] This disclosure also provides a novel antibody with advantageous properties such as manufacturability, stability, binding affinity, biological activity, targeting efficiency, reduced toxicity, an extended dose range that can be administered to patients, and potentially enhanced efficacy.

[0100] In one respect, an antibody is a complete antibody or an antibody fragment, such as Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, linear antibody, single-chain antibody molecule (e.g., scFv and scFab), single-domain antibody, or multispecific antibody formed from antibody fragments.

[0101] In one aspect, the antibody is capable of binding to CD19. In another aspect, the antibody is capable of binding to the same epitope on CD19 with any anti-CD19 antibody in the art (e.g., FMC63). In another aspect, the antibody is capable of binding to the same epitope on CD19 with a reference antibody comprising the amino acid sequence of SEQ ID NO: 21. In one aspect, the antibody comprises VH and VL, wherein VH comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; and VL comprises LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively.

[0102] In one aspect, the antibody comprises VH and VL, wherein VH comprises the amino acid sequence of SEQ ID NO: 1; and VL comprises the amino acid sequence of SEQ ID NO: 2. In one aspect, VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO: 1. In one aspect, VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO: 2. In one aspect, VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO: 2, wherein the amino acids at positions 39 and 80 of VL are R and P, respectively. In one aspect, VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, or 96% sequence identity with SEQ ID NO: 2, wherein the amino acids at positions 39, 80, 100, and 103 of VL are R, P, S, and R, respectively. In a specific embodiment, VH comprises or is composed of the amino acid sequence of SEQ ID NO: 1; VL comprises or is composed of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

[0103] In one aspect, VH and VL are linked via a flexible peptide linker. In another aspect, VH fuses with the C-terminus of VL via a flexible peptide linker through its N-terminus (VL-VH). In another aspect, VL fuses with the C-terminus of VH via a flexible peptide linker through its N-terminus (VH-VL). In one aspect, the flexible peptide linker is (G4S). n (SG4) n Or G4 (SG4) nWherein “n” is 1, 2, 3, 4, 5, 6, 7, 8, particularly 3. In one aspect, the flexible polypeptide linker comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20. In one aspect, the antibody comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In one aspect, the antibody comprises the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. The CDR and variable regions of exemplary antibodies are shown in columns 2-9 of Table 3.

[0104] In one aspect, this disclosure also provides a complex comprising an antibody and a heterologous molecule or portion thereof. In one aspect, the heterologous molecule or portion is covalently bound to the antibody. In one aspect, the complex is a fusion protein, such as a CAR. In one aspect, the complex comprises, from its N-terminus to its C-terminus, an extracellular antigen-binding domain comprising the antibody, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises the antibody. The extracellular antigen-binding domain, the transmembrane domain, and the intracellular signaling domain are described in Section II.

[0105] IV. Engineered Cells and Their Preparation

[0106] 4.1 Cells

[0107] In one aspect, this disclosure provides engineered cells comprising the aforementioned fusion protein, such as engineered immune cells. The engineered immune cells of this disclosure may be derived from a patient to be treated (i.e., autologous cells) or from a donor who is not a patient to be treated (e.g., allogeneic cells).

[0108] In one respect, cells are immune response cells. In another respect, cells are lymphoid lineage cells. In one respect, engineered immune cells are engineered T cells. In one respect, T cells originate from mammalian individuals. In another respect, T cells originate from primate individuals, such as human individuals. T cell and / or CD4+ and / or CD8+ T cell subtypes and subsets encompass naive T cells (TN cells), effector T cells (TEFF), memory T cells and their subtypes such as stem cell memory T cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM) or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T cells (MAIT cells), naturally occurring and adaptive regulatory T cells (Treg cells), helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and δ / γ T cells. Non-limiting examples of commercially available T cell lines include the cell line BCL2(AAA) Jurkat (ATCC). ® CRL-2902™), BCL2 (S70A) Jurkat (ATCC ® CRL-2900™), BCL2(S87A) Jurkat (ATCC ® CRL-2901™), BCL2 Jurkat (ATCC ® CRL-2899™), Neo Jurkat (ATCC ®CRL-2898™ and TALL-104 cytotoxic human T cell line (ATCC # CRL-11386). Other examples include, but are not limited to, mature T cell lines such as Deglis, EBT-8, HPB-MLp-W, HUT 78, HUT 102, Karpas 384, Ki225, My-La, Se-Ax, SKW-3, SMZ-1 and T34; and mature T cell lines such as ALL-SIL, Be13, CCRF-CEM, CML-T1, DND-41, DU.528, EU-9, HD-Mar, HPB-ALL, H-SB2, HT-1, JK-T1, Jurkat, Karpas 45, KE-37, KOPT-K1, K-T1, L-KAW, Loucy, MAT, MOLT-1, MOLT 3, MOLT-4, MOLT 13. MOLT-16, MT-1, MT-ALL, P12 / Ichikawa, Peer, PER0117, PER-255, PF-382, PFI-285, RPMI-8402, ST-4, SUP-T1 to T14, TALL-1, T ALL-101, TALL-103 / 2, TALL-104, TALL-105, TALL-106, TALL-107, TALL-197, TK-6, TLBR-1, TLBR-2, TLBR-3 and TLBR-4, CCRF-HSB-2 (CCL-120.1), J.RT3-T3.5 (ATCCTIB-153), J45.01 (ATCC CRL-1990), J.CaM1.6 (ATCC CRL-2063), RS4;11 (ATCC CRL-1873), CCRF-CEM (ATCC CRM-CCL-119); and cutaneous T-cell lymphoma lines, such as HuT78 (ATCC CRM-TIB-161), MJ[G11] (ATCC CRL-8294), and HuT102 (ATCC TIB-162). Non-limiting exemplary sources of such commercially available cell lines include the United States Type Culture Collection (ATCC) (Manassas, Virginia) and the German Collection of Microorganisms and Cell Cultures.

[0109] In some respects, the cell is a cytotoxic T cell (also known as TC, cytotoxic T lymphocyte, CTL, T killer cell, lytic T cell, CD8+ T cell, or killer T cell). In some respects, the T cell is a CD4+ T cell. In some respects, the T cell can be either a CD4+ T cell or a CD8+ T cell. In some respects, the cell is a tumor-specific T cell.

[0110] In one respect, the cells are natural killer (NK) cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells, tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer (LAK) cells, etc. NK cells can be isolated or obtained from commercially available sources. Non-limiting examples of commercially available NK cell lines include NK-92 (ATCC). ® CRL-2407™, NK-92MI (ATCC) ® CRL-2408™. Other examples include, but are not limited to, the NK cell lines HANK1, KHYG-1, NKL, NK-YS, NOI-90, and YT. Non-limiting exemplary sources of such commercially available cell lines include the American Type Culture Collection (ATCC) (Manassas, VA) and the German Collection of Microorganisms and Cell Cultures.

[0111] In one respect, the cells are B cells, monocytes or granulocytes, such as bone marrow cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils and / or basophils.

[0112] In some respects, the cells are myeloid cells. Non-limiting examples of myeloid cells include monocytes, macrophages, basophils, neutrophils, eosinophils, mast cells, erythrocytes, megakaryocytes, platelets, and stem cells that can differentiate into bone marrow cells. In some embodiments, the stem cells are pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells).

[0113] 4.2. Genetic Engineering Methods

[0114] In one aspect, engineered cells are prepared by various methods of transferring polynucleotides encoding fusion proteins (e.g., antigen receptors, such as CARs). Physical methods include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Biological methods include the use of DNA and RNA vectors, such as viral vectors, e.g., lentiviral vectors. Chemical methods include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, microbeads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary methods are shown in Table 4. In some aspects, those well-known methods include transduction via viruses (e.g., retroviruses or lentiviruses), transposons, and electroporation.

[0115] Table 4. Delivery methods used in genome editing systems

[0116] In one aspect, recombinant polynucleotides are transferred into cells using recombinant infectious viral particles (e.g., adenovirus vectors, AAV vectors, lentiviral vectors, retroviral vectors such as gamma-retroviral vectors). In one aspect, the retroviral or lentiviral vector has a long terminal repeat (LTR). In one aspect, the vector is self-inactivating (SIN). In one aspect, the vector is a conditionally replicative (mobile) vector. In one aspect, the lentiviral vector is derived from human, feline, or simian lentiviruses. In one aspect, the retroviral vector is derived from mouse retroviruses. In one aspect, lentiviruses or retroviruses include lentiviruses or retroviruses derived from any avian or mammalian cell source. In one aspect, lentiviruses or retroviruses are facultative, meaning they are capable of infecting host cells from several species, including humans. In one aspect, the gene to be expressed replaces the retroviral gag, pol, and / or env sequences.

[0117] In one aspect, the vector containing a polynucleotide encoding a fusion protein (e.g., a CAR) may contain a promoter and / or enhancer or regulatory element to regulate the expression of the encoded recombinant receptor. In one aspect, the promoter and / or enhancer or regulatory element may be a condition-dependent promoter, enhancer, and / or regulatory element. In one aspect, the polynucleotide encoding the fusion protein may be operatively linked to a constitutive promoter. In one aspect, the promoter is selected from cytomegalovirus (CMV) promoters, elongation factor-1α (EF1α) promoters, ubiquitin C (UbiC) promoters, glycerol phosphokinase (PGK) promoters, simian virus 40 (SV40) early promoters, and chicken β-actin promoters (CAGG) coupled to CMV early enhancers.

[0118] In one aspect, the polynucleotide is operatively linked to an inducible promoter. The inducible promoter can be induced by one or more conditions, such as physical conditions, the microenvironment of engineered immune effector cells, the physiological state of engineered immune effector cells, an inducer (i.e., an inducer), or a combination thereof. In one aspect, the inducing conditions do not induce the expression of endogenous genes in engineered mammalian cells and / or in individuals receiving the drug composition. In one aspect, the inducing conditions are selected from: inducers, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, tumor environment, and the activation state of engineered mammalian cells.

[0119] In one aspect, the polynucleotide is operatively linked to a post-transcriptional regulatory element (WPRE) of marmot hepatitis virus (WHP) located downstream of the polynucleotide.

[0120] In one aspect, the vector may contain a single promoter that drives the expression of one or more nucleic acid molecules. In another aspect, such nucleic acid molecules may be polycistronic. For example, in one aspect, the transcription unit may be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows co-expression of gene products (e.g., encoding a first CAR and a second CAR) via information from a single promoter. In one aspect, a single promoter may direct the expression of RNA containing two or three genes (e.g., encoding a first CAR and a second CAR) separated from each other by sequences encoding a self-cleaving peptide or a protease recognition site within a single open reading frame (ORF). In one aspect, the self-cleaving peptide is selected from foot-and-mouth disease virus (F2A), equine rhinitis virus (E2A), Thoseea asigna virus (T2A), and porcine swine chezinvirus-1 (P2A).

[0121] In one aspect, polynucleotides are transferred to T cells via electroporation. In another aspect, polynucleotides are transferred to T cells via transposition. In yet another aspect, polynucleotides are delivered via transposons, including the Sleeping Beauty transposon system (SB) and / or the piggyBac (PB) transposon system.

[0122] The polynucleotide encoding the fusion protein can bind to other coding regions encoding a secretory peptide or signal peptide that directs the secretion of the fusion protein. For example, if secretion of the fusion protein is desired, DNA encoding a signal sequence can be placed upstream of the fusion protein. Those skilled in the art will recognize that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or “mature” form of the polypeptide. In one aspect, the signal peptide comprises the sequence of a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, CD37, CD45, 4-1BB, GM-CSFR, IL-2, CD33, human IgKVIII, human IgG2 H, chymotrypsinogen, trypsinogen-2, HSA, insulin, or tPA signal peptide. Exemplary signal peptides comprise or consist of the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

[0123] In one aspect, the polynucleotide encoding the fusion protein contains a nucleic acid sequence encoding one or more markers. In another aspect, the one or more markers are transduction markers, alternative markers, and / or selection markers. In another aspect, the polynucleotide encoding the fusion protein contains a nucleic acid sequence encoding one or more additional fusion proteins that enhance and / or weaken cellular responses to adoptive transfer and ligand encounter.

[0124] In one aspect, the polynucleotide may also encode one or more alternative markers. In one aspect, alternative markers may include truncated forms of cell surface peptides, such as non-functional forms that do not transduce or cannot transduce signals, or signals normally transduced by the full-length form of the cell surface peptide, and / or truncated forms that are not internalized or cannot be internalized. In one aspect, truncated cell surface peptides include truncated forms of growth factors or other receptors, such as truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal growth factor receptor (EGFRt), prostate-specific membrane antigen (PSMA), or modified forms thereof. EGFRt can be used to identify or select cells engineered with EGFRt fusion proteins and their encoded exogenous proteins, and / or to remove or isolate cells expressing the encoded exogenous protein.

[0125] In one aspect, the biomarker is a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP) such as superfolded GFP (sfGFP), red fluorescent protein (RFP) such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed, or DsRed2, cyan fluorescent protein (CFP), blue-green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP) and their variants, including species variants, monomeric variants, and codon-optimized and / or enhanced variants of the fluorescent protein. In another aspect, the biomarker is or contains an enzyme, such as luciferase, the lacZ gene from *E. coli*, alkaline phosphatase, secretory embryonic alkaline phosphatase (SEAP), and chloramphenicol acetyltransferase (CAT). Exemplary luminescent reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or variants thereof.

[0126] In one aspect, the biomarker is a selection biomarker. In another aspect, the selection biomarker is a polypeptide that confers resistance to an exogenous active agent or drug. In another aspect, the selection biomarker is an antibiotic resistance gene. In another aspect, the selection biomarker is an antibiotic resistance gene that confers resistance to antibiotics in mammalian cells. In another aspect, the selection biomarker is selected from puromycin resistance genes, hygromycin resistance genes, blastomycin resistance genes, neomycin resistance genes, genimycin resistance genes, or bleomycin resistance genes or their modified forms.

[0127] Alternatively, the presence of recombinant DNA sequences in engineered cells can be confirmed using various assays, such as Southern and Northern blotting, RT-PCR, and PCR. In one aspect, fusion proteins can be detected by their ability to recognize target cells or by the release of cytokines such as interferon-γ, granulocyte / monocyte colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α), or interleukin-2 (IL-2). Furthermore, the function of fusion proteins can be evaluated by measuring cellular cytotoxicity.

[0128] 4.3 Preparation of engineered cells

[0129] In one aspect, this disclosure provides a method for manufacturing engineered cells. In another aspect, any known preparation method may be used. In a specific aspect, the method includes transducing an isolated cell population with a polynucleotide encoding a fusion protein and selecting said isolated cell subpopulation that has been successfully transduced with the polynucleotide, thereby producing genetically modified cells, as described above.

[0130] In one aspect, the method includes collection, isolation, transduction, and amplification steps. In a specific aspect, the method includes the following steps: (i) obtaining a population of immune cells (e.g., blood cells); (ii) isolating a specific cell population (e.g., T cells and / or NK cells); (iii) transducing the isolated cell population with a polynucleotide encoding a fusion protein; and (iv) amplifying the isolated cell subpopulation that has been successfully transduced with the nucleic acid sequence of step (iii), thereby producing genetically modified cells. These different steps are described in more detail below.

[0131] 4.3.1 Cell Collection

[0132] In one respect, the individual from whom the cells used to introduce the fusion protein (e.g., CAR) are obtained is an individual suffering from a disease or condition or requiring cell therapy or to whom cell therapy will be administered. In another respect, the cells may be derived from a healthy donor.

[0133] In one respect, cells may be obtained from a sample, such as a biological sample. In another respect, the sample is selected from whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor tissue, leukemia tissue, lymphoma tissue, lymph nodes, enteroassociated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsils, or other organs and / or cells derived therefrom. In one respect, the cells are primary cells. In another respect, cells are obtained from an individual's circulating blood, for example, through apheresis or leukapheresis. The resulting sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and / or platelets, and in some respects contains cells other than erythrocytes and platelets.

[0134] 4.3.2 Cell Isolation

[0135] There are various methods for conveniently isolating immune cells from samples, such as using Life Technologies Dynabeads. ® Systems; STEMcell Technologies EasySep™, RoboSep™, RosetteSep™, SepMate™; Miltenyi Biotec MACS™ Cell Isolation Kit; Cell Surface Marker Expression and other commercially available cell isolation and segregation kits (e.g., ISOCELL from Pierce, Rockford, IL). Specific immune cell subsets can be isolated by using microbeads or other binding agents targeting unique cell surface markers available in such kits. For example, MACS™ CD4+ and CD8+ microbeads can be used to isolate CD4+ and CD8+ T cells.

[0136] In one aspect, cell separation includes one or more affinity-based cell separation steps. In one aspect, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich desired components, lyse, or remove cells sensitive to a particular reagent. In one aspect, cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity, and / or resistance to a particular component.

[0137] In one approach, blood cells collected from an individual are washed, for example, to remove the plasma fraction and place the cells in a suitable buffer or medium for subsequent processing steps. In another approach, cells are washed with phosphate-buffered saline (PBS). In another approach, the wash solution is deficient in calcium and may be deficient in magnesium, or may lack many (if not all) divalent cations. An initial activation step in the absence of calcium can lead to amplified activation. In another approach, the washing step is performed using a semi-automatic flow-through centrifuge (e.g., the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In another approach, the washing step is performed using tangential flow filtration (TFF) according to the manufacturer's instructions. In another approach, after washing, the cells are resuspended in various biocompatible buffers (e.g., calcium-free buffers). 2+ / Mg 2+ In some embodiments, the blood cell sample is decomposed and the cells are resuspended directly in the culture medium. In one aspect, the separation includes density-based cell separation methods, such as preparing leukocytes from peripheral blood by lysing red blood cells and centrifuging with Percoll or Ficoll gradient.

[0138] In one aspect, the separation method includes separating different cell types based on the expression or presence of one or more specific molecules in the cells, such as surface markers, like surface proteins, intracellular markers, or nucleic acids. In another aspect, separation is based on affinity or immunoaffinity. Separation may be based on positive and / or negative selection, in which cells that have bound the reagent are retained for further use, and in negative selection, cells that have not bound the antibody or bound a partner are retained. Separation does not require 100% enrichment or removal of specific cell populations or cells expressing specific markers.

[0139] In one aspect, a separation step can remove cells that simultaneously express multiple markers, for example, by incubating cells with multiple antibodies or binding couplers, each specific for a marker targeted for negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating cells with multiple antibodies or binding couplers expressed on various cell types. In another aspect, multiple rounds of separation steps are performed, where portions of the positive or negative selection from one step are subjected to another separation step, such as subsequent positive or negative selection.

[0140] In some respects, specific T cell subsets, such as cells that are positive for or express high levels of one or more surface markers, are isolated using positive or negative selection techniques, including CD3+, CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells. In another respect, this is achieved through granules or microbeads conjugated to anti-CD3 / anti-CD28 (e.g., DYNABEADS). ® T cells are isolated by incubation with M-450 CD3 / CD28 T Cell Expander, MACSiBeads™, etc. In one embodiment, the positive selection time period is approximately 30 minutes. In another embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In one embodiment, the time period is 10 to 24 hours. In one embodiment, the incubation time is 24 hours. To isolate the desired cell population by positive or negative selection, the concentrations of cells and particles can be varied. In some embodiments, it may be necessary to significantly reduce the volume of the beads and cells mixed together (i.e., increase the cell concentration) to ensure maximum contact between cells and beads. In one embodiment, greater than 100 million cells / mL is used.

[0141] In some respects, T cells are isolated from a sample by negative selection of markers expressed on non-T cells such as B cells, monocytes, or other leukocytes such as CD14. In other respects, CD4 or CD8 selection steps are used to isolate CD4+ helper T cells and CD8+ cytotoxic T cells. This type of CD4+ and CD8+ population can be further sorted into subpopulations by positive or negative selection of markers expressed or expressed at relatively high levels on one or more naive, memory, and / or effector T cell subsets.

[0142] In some respects, for example, naive, central memory, effector memory, and / or central memory stem cells in CD8+ cells are further enriched or depleted through positive or negative selection based on surface antigens associated with the corresponding subsets. In some respects, central memory T (TCM) cells are enriched to improve efficacy, such as enhancing long-term survival, expansion, and / or engraftment after administration. In some respects, the combined use of TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

[0143] In some respects, memory T cells are present in the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. For example, using anti-CD8 and anti-CD62L antibodies, CD62L-CD8+ and / or CD62L+CD8+ fractions in PBMCs can be enriched or removed.

[0144] In some respects, the enrichment of central memory T (TCM) cells is based on the positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127. In some respects, it is based on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some respects, the CD8+ population of enriched TCM cells is separated by removing cells expressing CD4, CD14, and CD45RA and positively selecting or enriching cells expressing CD62L. In one respect, the enrichment of central memory T (TCM) cells begins with the negative fraction of cells selected based on CD4 expression, which undergoes negative selection based on CD14 and CD45RA expression and positive selection based on CD62L.

[0145] In one respect, NK cell enrichment is based on positive or high surface expression of CD56 and CD16, as well as negative expression of CD3 and / or optionally on the presence of NKp46 or NKp30 receptors.

[0146] In one aspect, a sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive materials, such as magnetically responsive particles or microparticles, like paramagnetic beads (e.g., Dynabeads). ® or MACS ® Microbeads. Magnetic-responsive materials (e.g., particles) are typically attached directly or indirectly to binding partners (e.g., antibodies) that specifically bind to molecules (e.g., surface markers) present on cells, multiple cells, or cell populations that require separation (e.g., for negative or positive selection). In one aspect, the sample is placed in a magnetic field, and those cells with attached magnetically responsive or magnetizable particles will attach to the magnetic beads and separate from unlabeled cells. For positive selection, cells attached to the magnetic beads are retained; for negative selection, unattached cells (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, wherein positive and negative portions are retained for further processing or undergo further separation steps.

[0147] In one aspect, magnetically responsive particles are attached to cells to be subsequently incubated, cultured, and / or engineered; in other aspects, the particles are attached to cells for administration to a patient. In one aspect, the magnetically responsive particles are removed from the cells. Methods for removing magnetically responsive particles from cells are known, including, for example, the use of competitive unlabeled antibodies, magnetically responsive particles, or antibodies conjugated to cleavable linkers. In one aspect, the magnetically responsive particles are biodegradable.

[0148] In one respect, affinity-based selection is achieved through magnetically activated cell sorting (MACS). ®(Performed at Miltenyi Biotec, Auburn, California). Magnetic activated cell sorting (MACS) was performed. ® The system is capable of selecting cells with magnetized particles attached to them with high purity. In some implementations, MACS... ® The process operates in a manner in which non-target and target substances are eluted sequentially after an external magnetic field is applied. That is, cells attached to the magnetized particles are held in place, while unattached substances are eluted. Then, after this first elution step is complete, the eluted substances are captured in the magnetic field and prevented from being released in a manner that allows them to be eluted and recovered. In some embodiments, non-target cells are tagged and removed from a heterogeneous cell population.

[0149] In one aspect, the cell population described herein is collected and enriched (or removed) by flow cytometry, wherein cells stained for multiple cell surface markers are carried in a fluid stream. In another aspect, the cell population described herein is collected and enriched (or removed) by preparative-scale sorting (FACS). In some embodiments, the cell population described herein is collected and enriched (or removed) by using a microelectromechanical system (MEMS) chip in conjunction with a FACS-based detection system. In both cases, cells can be labeled with multiple markers, allowing for the separation of specific T cell subsets with high purity.

[0150] In one aspect, the preparation method includes the step of freezing (e.g., cryopreservation) cells before or after isolation, incubation, and / or engineering. In one aspect, the freezing and subsequent thawing steps remove granulocytes and monocytes from the cell population to some extent. In one aspect, for example after a washing step, the cells are suspended in a freezing solution to remove plasma and platelets. In some aspects, any of a variety of known freezing solutions and parameters can be used. One example involves using PBS or other suitable cell freezing media containing 20% ​​DMSO and 8% human serum albumin (HSA). This is then diluted 1:1 with the medium so that the final concentrations of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to -80°C at a rate of 1°C / min and stored in the gas phase of a liquid nitrogen storage tank. In one aspect, the cryopreserved cells are thawed and washed as described herein and allowed to stand at room temperature for 1 hour before activation.

[0151] 4.3.3 Cell Expansion

[0152] In one aspect, the provided method includes steps of cultivation, incubation, culturing, and / or genetic engineering. In one aspect, cells are incubated and / or cultured prior to or in connection with genetic engineering. The incubation step may include cultivation, culturing, stimulation, activation, and / or proliferation. In one aspect, cells are incubated under stimulating conditions or in the presence of a stimulant. The conditions may include one or more of a specific culture medium, temperature, oxygen content, carbon dioxide content, time, active agents (e.g., nutrients, amino acids, antibiotics, ions) and / or stimulating factors (e.g., cytokines, chemokines, antigens, binding couplers, fusion proteins, recombinant soluble receptors) and any other active agents designed to activate the cells.

[0153] In one aspect, the stimulating condition or stimulant includes one or more active agents, such as ligands, capable of stimulating or activating intracellular signaling domains of the TCR complex. In some aspects, the active agent initiates or activates the TCR / CD3 intracellular signaling cascade in T cells. Such active agents may include antibodies, such as those specific to TCR components and / or co-stimulatory receptors (e.g., anti-CD3, anti-CD28), for example, bound to solid supports such as microbeads (e.g., Dynabeads). ® ) and / or one or more cytokines. In one aspect, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, 50 million, 75 million, 80 million, 85 million, 90 million, 95 million, 100 million, 125 million, or 150 million cells / mL are used.

[0154] In one embodiment, the mixture can be cultured for several hours (about 3 hours) to about 14 days or any integer value of hours in between. In another embodiment, the mixture can be cultured for 21 days. In one embodiment, the microbeads and T cells are cultured together for about 8 days. In another embodiment, the microbeads and cells are cultured together for 2-3 days. Several stimulation cycles may also be required, so that the culture time of T cells can be 60 days or longer. Suitable conditions for T cell culture include appropriate culture media containing factors necessary for proliferation and viability, including interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives. Other additives for cell growth include, but are not limited to, surfactants, plasma enzymes, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Culture media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, optimizers, added amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or some cytokines sufficient to grow and expand T cells. Antibiotics (e.g., penicillin and streptomycin) are included only in the experimental culture and not in the cell culture to be infused into individuals. Target cells are maintained under conditions necessary to support growth, such as a suitable temperature (e.g., 37°C) and atmosphere (e.g., air with 5% CO2).

[0155] In one aspect, NK cells are expanded in vitro using interleukin-2 (IL-2), IL-15, the IL-15 / IL-15RA complex, IL-18, and IL-12. In another aspect, the NK cells are expanded in vitro for at least about 5 days prior to administration to a patient, for example, not less than about 10 days, not less than about 15 days, or not less than about 20 days.

[0156] V. Pharmaceutical Composition

[0157] The pharmaceutical compositions disclosed herein may comprise engineered cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise: buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In one aspect, the compositions of this disclosure are formulated for intravenous administration.

[0158] In one aspect, the pharmaceutical composition is substantially free of, for example, detectable levels of contaminants, such as endotoxins, mycoplasma, replicative lentiviruses (RCL), p24, VSV-G nucleic acids, HIV gag, residual anti-CD3 / anti-CD28 coated microbeads, mouse antibodies, mixed human serum, bovine serum albumin, bovine serum, culture medium components, vector packaging cell or plasmid components, and the group consisting of bacteria and fungi. In one aspect, the bacteria are selected from Alcaligenes faecalis (Alcaligenes comatus). Alcaligenes faecalis Candida albicans ( Candida albicans ), Escherichia coli ( Escherichia coli Haemophilus influenzae ( ) Haemophilus influenza ), Neisseria meningitidis ( Neisseria meningitides ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa Staphylococcus aureus ( Staphylococcus aureus Streptococcus pneumoniae () Streptococcus pneumonia ) and Group A Streptococcus pyogenes ( Streptococcus pyogenes group A At least one of the groups consisting of ).

[0159] When indicated as "immunely effective dose," "antitumor effective dose," "tumor-suppressive effective dose," or "therapeutic dose," the precise amount of the composition of this disclosure to be administered can be determined by a physician taking into account individual differences in the patient's (individual's) age, weight, tumor size, degree and condition of infection or metastasis. Generally, a pharmaceutical composition containing T cells described herein can be administered in doses of 10... 4 Up to 10 9 Cells / kg body weight, in some cases 10 5 Up to 10 6 Administer at a dose of cells per kg body weight (including all integer values ​​within those ranges). T-cell compositions may also be administered at these doses once or multiple times. Cells can be administered using infusion techniques commonly known in immunotherapy.

[0160] VI. Treatment Methods

[0161] The pharmaceutical compositions disclosed herein can be administered in a manner suitable for treating (or preventing) a disease. Such diseases may include solid tumors, such as sarcomas and carcinomas, including fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovial tumors, mesotheliomas, Ewing's tumors, leiomyosarcomas, rhabdomyosarcomas, colon cancer, lymphomas, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, and Wilms' tumor. Tumors, including cervical cancer, testicular tumors, seminoma, bladder cancer, melanoma, and CNS tumors (such as gliomas (e.g., brainstem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germ cell tumor, medulloblastoma, Schwannoma, craniopharyngioma, ependymoma, pineal tumor, angioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases); and non-solid tumors such as leukemia, including acute... Leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and spinal dysplasia.

[0162] The dosage and frequency of administration will be determined by the patient's condition and the type and severity of their disease, although the appropriate dosage may be determined through clinical trials. The subject composition can be administered in any convenient manner, including via aerosol injection, ingestion, infusion, implantation, or transplantation. The compositions described herein can be administered to patients via arterial, subcutaneous, intradermal, intratumoral, intralymphatic, intramedullary, intramuscular, intravenous (iv), or intraperitoneal injection. In one aspect, the T-cell compositions of the present invention are administered to patients via intradermal or subcutaneous injection. In another aspect, the composition is injected directly into the target organ (e.g., an organ affected by a tumor). Alternatively, the composition may be indirectly delivered to the target organ, for example, by administration to the circulatory system (e.g., the tumor vascular system). Amplification and differentiation agents may be provided before, during, or after the administration of cells or the composition to increase the production of T cells or NK cells in vitro or in vivo.

[0163] In one aspect, for example, lymphodepletion is performed on an individual prior to the administration of one or more of the cells described herein. In one aspect, lymphodepletion includes the administration of one or more of melphalan, cytoxan, cyclophosphamide, and fludarabine.

[0164] In one aspect, engineered cells are administered as part of a combination therapy, such as simultaneously or sequentially with another therapeutic intervention (e.g., an antibody or engineered cell or receptor or active agent, such as a cytotoxic agent or therapeutic agent). In some embodiments, cells or antibodies are administered co-administered with one or more additional therapeutic agents or in combination with another therapeutic intervention, simultaneously or sequentially in any order. In one aspect, cells are co-administered with another therapy at sufficiently close times such that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. In one aspect, cells or antibodies are administered prior to one or more additional therapeutic agents. In one aspect, cells or antibodies are administered after one or more additional therapeutic agents, such as an anticancer agent. In the context of this disclosure, cell therapy is contemplated for use in similar combinations with chemotherapy agents, radiotherapy agents, or immunotherapy interventions, as well as apoptosis-promoting or cell cycle regulators (such as immune checkpoint inhibitors).

[0165] Alternatively, the therapy of this application may be administered before or after other active agent treatments (ranging from minutes to weeks). In some embodiments, the other active agent and the cells or antibodies of this disclosure are administered to the individual separately, generally ensuring that there is no significantly excessively long time interval between each delivery so that the active agent and the therapy will still be able to exert a beneficial combined effect on the cells. In this case, it is anticipated that the cells can be contacted with two administration methods at intervals of approximately 12-24 hours, more preferably approximately 6-12 hours. However, in some cases, it may be necessary to significantly extend the treatment period, with intervals of several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) between each administration. If necessary, the treatment cycle is expected to be repeated. It is also anticipated that various standard therapies as well as surgical interventions can be used in combination with cell therapy.

[0166] VII. Sequence

[0167] Table 5. Sequences of this disclosure

[0168] VIII. Examples

[0169] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0170] Recombinant DNA technology

[0171] Manipulate DNA using standard methods, as described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*; Cold Spring Harbor Laboratory Press, New York, 1989. Use molecular biology reagents according to the manufacturer's instructions. For general information on the nucleotide sequences of the light and heavy chains of human immunoglobulins, see: Kabat, E.A. et al., (1991) *Sequences of Proteins of Immunological Interest*, 5th ed., NIH Publication No. 91-3242.

[0172] DNA sequencing

[0173] DNA sequences were determined using double-strand sequencing.

[0174] Gene synthesis

[0175] If needed, the required gene fragments can be generated by PCR using a suitable template, or synthesized from synthetic oligonucleotides and PCR products via automated gene synthesis. Gene fragments flanked by a single restriction endonuclease cleavage site are cloned into standard cloning / sequencing vectors. Plasmid DNA is purified from transformed bacteria and its concentration is determined by UV spectroscopy. The DNA sequence of the subcloned gene fragment is confirmed by DNA sequencing. Gene fragments are designed with suitable restriction sites to allow subcloning into their respective expression vectors. All constructs are designed with a 5'-terminal DNA sequence encoding a leader peptide that targets a protein for secretion in eukaryotic cells.

[0176] Protein expression

[0177] Proteins are expressed in host cells for several days as needed. Standard cell culture techniques are used, as described in Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harford, JB, Lippincott-Schwartz, J., and Yamada, KM (eds.), John Wiley & Sons, Inc.

[0178] Protein purification

[0179] Proteins were purified from filtered cell culture supernatant following a standard protocol. Briefly, antibodies were applied to a Protein A Sepharose column and washed with PBS. The antibodies were eluted at low pH, and the sample was then immediately neutralized.

[0180] Example 1: Generation and evaluation of anti-CD19 antibodies

[0181] Antibodies containing FMC63 scFv, scFv-1, scFv-2 or scFv-3 (sequences shown in Table 3) fused with human IgG1Fc fragments were synthesized, and their affinity for the homologous ligand CD19 was detected by surface plasmon resonance (SPR).

[0182] All SPR experiments were performed on a Biacore T200 (PCytiva) using HBS-EP as the run buffer. Antibodies were captured via a protein A chip (LOT# 10309880, Cytiva). Soluble CD19 protein (amino acids 20-291) was used as the analyte at concentrations ranging from 300 nM to 4.69 nM, flowd through a flow cell for 120 seconds, followed by dissociation analysis for 300 seconds. The binding rate (Kon), dissociation rate (Koff), and constant Ka (Kon / Koff) were fitted to the results. As shown in Figure 1, antibodies containing scFv-1 and scFv-2 exhibited enhanced affinity compared to other binding agents.

[0183] Example 2: Generation and Evaluation of CD19 CAR

[0184] The CARs in Table 6 are formed by fusing a signal peptide (signal peptide-1 of CAR-W, signal peptide-2 of CAR-1 to CAR-13) with FMC63scFv, scFv-1, scFv-2, or scFv-3, followed by four distinct spacer regions, a transmembrane region derived from human CD28, and an intracellular domain encoding signal motifs from 41BB and CD3ζ. Each CAR is co-expressed with a truncated EGFR (EGFRt, sequence shown in SEQ ID NO: 40) as a transduction marker. Both the CAR and EGFRt are encoded by a single promoter construct isolated by self-cleavage of the T2A peptide.

[0185] Table 6. Structures of candidate CARs (from N-terminus to C-terminus)

[0186] Primary human T cell populations expressing various CARs were generated. Nucleic acid molecules encoding each fusion protein were cloned into lentiviral vectors. T cells isolated from human PBMC samples obtained from healthy donors were processed using CD3 / CD28 microbeads (DYNABEAD). ® Stimulation. One day after stimulation, cells were transduced using a lentiviral vector and then expanded for 6 days. Cells were treated with EGFR-AF488 and CD19. pro - APC staining and detection by flow cytometry. CD19 binding capacity was used as a surrogate indicator of CAR stability and affinity and was examined by analyzing the mean fluorescence index (MFI) from CD19 staining after gating EGFRt-positive cells.

[0187] Compared to other spacer regions carrying the same binding agent, CARs containing spacer region S-3 (CAR-3, CAR-7, and CAR-11) have an increased ability to bind CD19. Figure 2 CAR-3 showed the highest binding capacity.

[0188] Example 3: Substrate signal transduction of CD19CAR construct

[0189] Some CARs can exhibit antigen-independent activity or signaling, also known as basal signaling. Excessive basal signaling can lead to increased differentiation and T cell exhaustion. To assess basal signaling in the transduced CAR T cells of Example 2, cells were co-cultured for 8 days in the absence of exogenous stimulation. IFNγ concentrations were then measured using the Perkin Elmer AlphaLISA assay kit according to the manufacturer's instructions.

[0190] exist Figure 3 Among them, CARs containing the S-3 spacer region (CAR-3, CAR-7 and CAR-11) exhibited low levels of basal signaling, as indicated by low levels of antigen-independent IFNγ secretion.

[0191] Example 4: Kill efficiency and CD69 expression of CD19 CAR construct

[0192] Primary human T cells from four healthy donors were transduced using lentiviral vectors containing polynucleotides encoding the fusion proteins CAR-W, CAR-3, CAR-7, or CAR-11 described in Example 2. The transduction efficiency (assessed by EGFR staining) of all samples was normalized to 50% by adding non-transduced cells. Cytotoxicity was then examined after challenging these transduced cells with Raji target cells engineered to express approximately 6,704 copies or 74,602 copies of CD19 on their cell surface. Figure 4AEffector cells or control cells expressing only EGFRt were co-cultured with target cells at a 1:1 E:T ratio. After 20 hours, cells were isolated and stained with CD3, EGFR, CD20, and CD69.

[0193] The killing efficiency of each CAR T cell sample was calculated as the percentage of remaining target cells co-cultured with T cells transduced only with EGFRt control. For example... Figure 4B As shown, CAR-3 and CAR-11 showed higher killing efficiency than observed for CAR-W. All transduced CAR T cells showed comparable levels of CD69 expression ( Figure 4C ).

[0194] Example 5: Cytokine release and T cell expansion after target cell stimulation

[0195] Cytokine release was assessed using effector cells from Example 4. Supernatants from Example 4 were collected and the accumulated IFN-γ, TNF-α, and IL-2 cytokines were analyzed. Results are shown in… Figures 5A-5C In the study, cells transduced with CAR-3, CAR-7, and CAR-11 showed similar secretion of IFNγ and TNFα after being attacked by tumor cells. However, CAR-3 and CAR-7 produced more IL-2.

[0196] In the extended assay of Example 4, T cells were proliferated for 7 days after initial stimulation with target cells, and the number of CART cells was assessed. Figure 5D As shown, T cells transduced with CAR-3 and CAR-7 exhibited significantly higher T cell numbers compared to other CAR constructs.

[0197] Example 6: Kill efficiency of CD19 CAR at different E:T ratios

[0198] Primary human T cells from three healthy donors were transduced using lentiviral vectors (effector cells) containing polynucleotides encoding the fusion proteins CAR-W or CAR-13 from Examples 1-2, or lentiviral vectors containing polynucleotides encoding EGFRt. The transduction efficiency (assessed by EGFR staining) of all samples was normalized to 50% by adding non-transduced cells. Cells were then transduced using Raji-CD19. Lo Cytotoxicity was examined after target cells attacked these effector cells at various E:T ratios. Effector cells or control cells expressing only EGFRt were compared with Raji-CD19. Lo Target cells were co-cultured at E:T ratios of 1:1, 1:2, and 1:8. On day 3 of co-culture assays, cell samples were isolated and stained with CD3, EGFR, and CD20.

[0199] The killing efficiency of each CAR-T cell sample was calculated as the percentage of remaining target cells co-cultured with T cells transduced only with EGFRt. For example... Figure 6 As shown, the kill efficiency of both CAR-W and CAR-13 decreases with increasing E:T ratio. However, at E:T ratios of 1:2 (p=0.0129, CAR-13 vs. CAR-W) and 1:8 (p=0.0367, CAR-13 vs. CAR-W), CAR-13 showed better performance than CAR-W against Raji-CD19. Lo The elimination of target cells was greater. This data demonstrates the enhanced anti-tumor activity of CAR-T cells through the S-3 spacer region.

Claims

1. A fusion protein comprising a) Extracellular antigen-binding domain; b) A spacer region consisting of the amino acid sequence of SEQ ID NO: 28 or its functional equivalent; c) Transmembrane domains; and d) Intracellular signal transduction domains.

2. The fusion protein according to claim 1, wherein the spacer region is composed of the amino acid sequence of SEQ ID NO: 31 (EX1KSCDTPPPX2PX3CP); wherein X1 is S or P; X2 is C or S; and X3 is R or P; Preferably, the spacer region consists of the amino acid sequence of SEQ ID NO: 31 (EX1KSCDTPPPX2PX3CP); wherein X1 is S; X2 is C or S; and X3 is R or P; More preferably, the spacer region consists of the amino acid sequence of SEQ ID NO:

28.

3. The fusion protein according to claim 1 or 2, wherein the extracellular antigen-binding domain comprises an antibody, and the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL). Preferably, the antibody is capable of binding to the same epitope on CD19 with a reference antibody containing the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 25; More preferably, the VH comprises HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; the VL comprises LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively; or The VH comprises HCDR1, HCDR2, and HCDR3 containing the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively; the VL comprises LCDR1, LCDR2, and LCDR3 containing the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

4. The fusion protein according to claim 3, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1; and the VL comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2, wherein the amino acids at positions 39 and 80 of the VL are R and P, respectively. Preferably, the VL contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 2, and optionally the amino acids at positions 100 and 103 of the VL are S and R, respectively; More preferably, the VH contains the amino acid sequence of SEQ ID NO: 1; and the VL contains the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:

4.

5. The fusion protein according to claim 3 or 4, wherein the antibody is scFv, and the VH is fused to the C-terminus of the VL via a flexible polypeptide linker at its N-terminus; Preferably, the flexible polypeptide linker comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20; More preferably, the scFv contains the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO:

23.

6. The fusion protein according to any one of claims 1 to 5, wherein the transmembrane domain comprises a transmembrane domain of CD8α, CD4, CD28, CD137, CD80, CD86, CD152 or PD1; Preferably, the transmembrane domain comprises a CD8α, CD4, or CD28 transmembrane domain; More preferably, the transmembrane domain comprises the amino acid sequence of SEQ ID NO:

32.

7. The fusion protein according to any one of claims 1 to 6, wherein the intracellular signal transduction domain comprises a co-stimulatory signal transduction domain; Preferably, the co-stimulatory signal transduction domain is selected from the signal transduction domains of ligands and combinations thereof of CD27, CD28, CD137, OX40, CD30, CD40, CD3, HVEM, ICOS, Myd88, LFA-1, ICOS, CD2, CD7, NKG2C, B7-H3, and CD83. More preferably, the co-stimulatory signal transduction domain is a CD28 or CD137 signal transduction domain.

8. The fusion protein according to any one of claims 1 to 7, wherein the intracellular signal transduction domain comprises a primary intracellular signal transduction domain; Preferably, the primary intracellular signal transduction domain includes the cytoplasmic signal transduction domain of CD3ζ; More preferably, the intracellular signal transduction domain includes a co-stimulatory signal transduction domain fused to the N-terminus of a primary intracellular signal transduction domain via its C-terminus, wherein the co-stimulatory signal transduction domain is a CD137 signal transduction domain; and the primary intracellular signal transduction domain includes a CD3ζ cytoplasmic signal transduction domain.

9. The fusion protein according to any one of claims 1 to 8, wherein the fusion protein is a CAR, and the CAR comprises, from its N-terminus to its C-terminus, the following in sequence: an extracellular antigen-binding domain, a spacer region, a transmembrane domain, and an intracellular signal transduction domain; Preferably, the extracellular antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23, and the spacer region is composed of the amino acid sequence of SEQ ID NO: 28; More preferably, the CAR contains the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO:

36.

10. An antibody that binds to CD19, comprising VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1; and the VL comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 2, wherein the amino acids at positions 39 and 80 of the VL are R and P, respectively. Preferably, the VL contains an amino acid sequence that is at least 95% identical to SEQ ID NO: 2, wherein the amino acids at positions 39 and 80 of the VL are R and P, respectively; and optionally, the amino acids at positions 100 and 103 of the VL are S and R, respectively. More preferably, the VH contains the amino acid sequence of SEQ ID NO: 1; and the VL contains the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:

4.

11. The antibody of claim 10, wherein the antibody is scFv, and the VH is fused to the C-terminus of the VL via a flexible polypeptide linker at its N-terminus; Preferably, the flexible polypeptide linker comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20; More preferably, the antibody comprises the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO:

23.

12. A complex comprising the antibody and a heterologous molecule or portion thereof as described in claim 10 or 11; Preferably, the heterologous molecule or part thereof is covalently bound to the antibody; More preferably, the complex is a fusion protein, which comprises, from its N-terminus to its C-terminus, an extracellular antigen-binding domain containing the antibody, a transmembrane domain, and an intracellular signal transduction domain.

13. An isolated nucleic acid encoding a fusion protein according to any one of claims 1 to 9 or an antibody according to claim 10 or 11.

14. A vector comprising the nucleic acid of claim 13, preferably a viral vector, more preferably a retrovirus, lentivirus, adenovirus or adeno-associated virus vector.

15. A cell comprising the fusion protein of any one of claims 1 to 9, the antibody of claim 10 or 11, the complex of claim 12, the isolated nucleic acid of claim 13, or the vector of claim 14, preferably, the cell being a lymphocyte, more preferably, the cell being an NK cell or a T cell.

16. A pharmaceutical formulation comprising the fusion protein of any one of claims 1 to 9, the antibody of claim 10 or 11, the complex of claim 12, or the cell and pharmaceutically acceptable carrier of claim 15.

17. A method of treating cancer or an autoimmune disease in an individual, comprising administering to the individual the fusion protein of any one of claims 1 to 9, the antibody of claim 10 or 11, the complex of claim 12, or the cell of claim 15. Preferably, the cancer is selected from leukemia, lymphoma, lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, and rhabdomyosarcoma; the autoimmune disease is selected from systemic lupus erythematosus, lupus nephritis, multiple sclerosis, rheumatoid arthritis, Sjögren's syndrome, idiopathic thrombocytopenic purpura, type 1 diabetes, pemphigus vulgaris, neuromyelitis optica, ANCA vasculitis, and myasthenia gravis. More preferably, the cancer is leukemia or lymphoma; the autoimmune disease is systemic lupus erythematosus or lupus nephritis.

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